The peripheral membrane protein, EEA1, has recently been defined as an essential participant in the process of early endosome fusion. EEAI has been postulated to work as an effector of a small GTPase, RabS, which has been known to be essential in the control of fusion. EEAI also interacts directly with the PI 3-kinase product PI3P. The interaction of EEAI with these molecules occurs through motifs in its COOH terminus. One of these is a double Zn++ finger called FYVE, which directly interacts with PI3P. Immediately adjacent to this domain is a stretch of 20 amino acids, which are necessary for RabS binding. In addition, immediately adjacent to the RabS binding region is an IQ motif, which represents a consensus sequence for calmodulin (CaM) binding. We wish to elucidate the precise function of EEAI in endosome fusion, and in particular the role that these three closely spaced structural motifs play in determining the membrane topography and function of EEAI. We propose the following Specific Aims: SPECIFIC AIM 1. We will test the hypothesis that Ca++/CaM interacts directly with EEAI and that this interaction is functionally important for the fusion step. SPECIFIC AIM 2. We will test the hypothesis that the binding of the three known C-terminal ligands of EEAI (RabS, CaM and PtdIns3P) is interdependent, and functionally relevant in the temporal control of fusion. SPECIFIC AIM 3. We will test the hypothesis that the interaction of EEAI with RabS influences the kinetics of the GTP cycle of RabS, as a mechanism to limit the extent of fusion. SPECIFIC AIM 4. We will investigate the topology and dynamics of EEAI and its ligands in live cells. For this, we will construct EEAI molecules containing mutations in critical elements of the RabS, PtdIns3P and calmodulin interaction motifs, and determine their effects on the dynamics of endosome tethering and fusion in intact live cells using high speed digital imaging microscopy.